Next-Generation Sequencing Services
Next-Generation Sequencing Services
We offer multiple options for DNA sequencing, including whole genome, whole exome, and targeted sequencing.
DNA Submission Guidelines
Standard DNA protocols require a minimum of 200ng of DNA. Upon submission, all DNA concentrations will be checked via Qubit and quality will be assayed via Agilent Tapestation or Bioanalyzer. Core staff will send you the QC results and can advise you on suitability for downstream NGS applications. We also offer options for prepping low input, high molecular weight, or degraded (e.g. from FFPE samples) DNA.
Whole Genome Sequencing (WGS)
For WGS, the core offers different options, including standard DNA, PCR-Free, and linked-reads. Standard DNA preps are ideal for large genome sequencing when the amount of available DNA is limited. The PCR-Free option enhances the ability to sequence through more challenging genomic regions but requires at least 1ug of intact genomic DNA.
Whole Exome Sequencing (WES)
Whole-exome sequencing uses biotinylated oligonucleotide probes to capture only the protein coding regions (gene exons) from genomic DNA library. Unlike WGS, only the protein coding genes are sequenced thereby reducing the amount of sequence needed per sample. Our preferred WES option uses the IDT Exome Research Panel, but we do work with other probe sets when requested.
Targeted sequencing is used when deep sequencing depth over specific genomic regions is desired. There are two methodologies for approaching targeted sequencing assays: probe-based capture sequencing or amplicon sequencing. We can help you select a commercially available panel or design a custom solution to interrogate your genes/regions of interest. Please contact the core for more details.
Linked-Read Libraries utilize the 10X Chromium microfluidic system to partition and barcode HMW DNA prior to building an Illumina compatible DNA library. Long Ranger analysis pipelines deconvolutes the HMW barcodes to stitch the short reads from the sequencer together, thereby allowing for phased and/or structural variant detection.
Although we do not perform ChIP or 3C/4C/5C/HiC assays in the core, we do prepare the Illumina libraries from DNA obtained from these and other protocols. Please contact the core to inquire about prep options.
RNA Sequencing Libraries
We provide a large array of RNA sequencing services, including poly-A enrichment, ribo-depletion, 3′-mRNA counting (QuantSeq), and small RNA library preps. Please note that by default ALL of our library preps are strand-specific.
RNA Submission Guidelines
Standard RNA protocols require a minimum of 500ng of total RNA. RNA must be DNAse treated to remove contaminating DNA prior to submission. RNA quality will be checked via Agilent Tapestation or Bioanalyzer. Core staff will send you the QC results and can advise you on suitability for downstream NGS applications. For samples not meeting these minimum requirements, we also offer low input and/or degraded prep options that tolerate either lower input (<100ng) or highly degraded (RIN<5) RNA.
Messenger RNA accounts for <5% of the RNA in cells. Thus, for transcriptome or gene expression studies, an enrichment of samples for mRNAs is preferable. Poly-A selection, in which poly-adenylated transcripts are selected via hybridization to poly-T oligos bound to magnetic beads, is the most commonly used method for enriching mRNA from total RNA samples. For low input (<200ng) samples, the Smart-Seq method starts by hybridizing the RNA to an oligo(dT)-containing primer and then uses a template switching mechanism to generate full-length cDNA. Both of these polyA-dependent methods generate the highest percentage of reads mapping to protein encoding genes and thus are the first choice for many investigators. Poly-A enrichment requires high-quality total RNA samples so it not suitable for FFPE or otherwise degraded samples. For human/animal samples, a RIN>7 is recommended.
Ribosomal RNA depletion is applied when transcripts do not carry polyA (e.g. bacterial RNA), when you want to characterize long non-coding RNA (lncRNA) as well as mRNA, or when your RNA is degraded. There are two methodologies for removing ribosomal RNAs (rRNA) from your total RNA. The more common option is a subtractive hybridization method. Suitable for samples with RINs>5, this method uses oligonucleotide probes and magnetic beads to capture and remove the rRNA from the sample. More appropriate for degraded samples (RINs<5) is the RNaseH method which enzymatically degrades rRNA targeted by complementary oligonucleotides.
QuantSeq (3′-mRNA Counting)
QuantSeq provides cost-efficient, gene expression profiling data without the need for polyA-enrichment or rRNA depletion. Reads are generated towards the poly(A) tail and directly correspond to the mRNA sequence simplifying the bioinformatic analysis. QuantSeq is an excellent alternative to microarrays.
Due to the size, small RNA is lost during most library prep procedures. For small (micro) RNA, we offer library construction using the NEBNext Small RNA kit from New England BioLabs. Small RNA preps can start from either enriched small RNA or directly from total RNA (RIN>7). The minimum amount of total RNA is 100ng, but we recommend starting 500ng-1000ng if possible as the fraction of small RNA in a sample can vary between tissues/organisms.